作　　者： ; ; ;

机构地区： 重庆大学生物工程学院生物流变科学与技术教育部重点实验室

出　　处： 《生物化学与生物物理进展》 2010年第3期326-336,共11页

摘　　要： 轻链钙调蛋白结合蛋白(light-chain caldesmon,l-CaD)是一种肌动蛋白结合蛋白,它通过与肌动蛋白结合而稳定胞内微丝结构,在磷酸化作用下则能从微丝上脱离.在众多非转移性癌细胞以及永生化的正常细胞系中,l-CaD的表达量很低甚至没有,但在高迁移活性的转移性癌细胞中,l-CaD表达量显著上升,因此l-CaD可能是维持转移性癌细胞高迁移能力的重要因素.为了探索l-CaD如何调节转移性癌细胞迁移活性及其所处地位,以人源转移性乳腺癌细胞MDAMB-231作为载体,一方面,在胞内高表达外源野生型l-CaD及其磷酸化突变株,干扰胞内l-CaD的磷酸化进程,从而考察l-CaD磷酸化对细胞迁移的调节,另一方面,利用siRNA技术,抑制l-CaD在MDAMB-231细胞内的表达量,检测l-CaD对转移性癌细胞迁移活性的总体影响.通过细胞骨架荧光染色、细胞迁移小室、单细胞层次上的牵张力测定以及细胞基底脱黏附能力测定,结果显示:a.阻断MDAMB-231胞内l-CaD的磷酸化进程将显著抑制细胞的迁移能力,细胞骨架调整受阻,基底牵张力增加,细胞基底脱附能力下降;b.l-CaD表达抑制的MDAMB-231细胞失去了完整的细胞骨架,其迁移能力显著降低并与非转移性癌细胞MCF-7类似,而细胞骨架解聚导致MDAMB-231细胞基底牵张力显著减少,更容易从基底脱附.总的来说,l-CaD磷酸化是调节转移性癌细胞高迁移活性的重要分子开关,l-CaD的高表达与高效磷酸化循环是维持转移性癌细胞高迁移活性的关键因素.探索了l-CaD调节转移性癌细胞迁移活性的机制,也为认识转移性癌细胞的本质提供了新的思路. The light-chain actin-binding protein caldesmon （l-CaD） stabilizes microfilaments and stress fibers in cells, it also can dissociate from the actin filament by phosphorylation. Curiously, in many tumor and transformed cells CaD is down-regulated, but in metastatic cancer lines, the expression of l-CaD is very high, in order to explore the role of l-CaD in metastatic cancer cell mobility, transwell migration assays and contractility measurements at cellular levels by traction microscopy were performed using metastatic human breast cancer cell lines the MDA MB-231.By over-expression of wild-type or mutated CaD, including A1234 （unphosphorylatable, both PAK- and ERK-sites converted to Ala） and D1234 （phosphorylation mimics, both PAK- and ERK-sites converted to Asp） in MDA MB-231 cells, l-CaD phosphorylation in cells was disturbed to detect how the l-CaD phosphorylation mediate cancer cell migration. Afterwards, by siRNA , the l-CaD expression in MDA MB-231cells was knocked down and the migration activities was compared with the non-metastatic human breast cancer cell line MCF-7. The result showed that the A1234 mutant cells exhibited most robust traction force and the wild-type CaD transfected cells also showed much stronger traction force than control cells, whereas the same transfection resulted in most severely hampered migration in both types of cells. These A1234 and wild-type expressing cells also exhibited enhanced stress fibers and delayed trypsin-induced detachment from substratum. Upon the inhibition of the CaD expression, the l-CaD knock-down cells lost the stress fiber structures and exhibited significant decrease in migration activities which was similar to the non-metastatic cancer cell line MCF-7. Moreover, because of the disruption of the cytoskeleton by l-CaD knock-down, the contractility of the l-CaD knock-down cells was decreased and much easier to detach from the substratum. Taken together, l-CaD phosphorylation is an important pathway that mediate the migration activity of the metast

关 键 词： 钙调蛋白结合蛋白 细胞骨架 转移性癌细胞 细胞迁移 细胞基底牵张力

领　　域： [生物学] [生物学]